1. Field of the Invention
This invention relates to specific binding assays in which binding proteins specific for a sample analyte are bound by antibodies. The antibodies are specific for different epitopes of the binding protein and can be attached to either soluble or insoluble materials which facilitate separation procedures.
2. Description of the Prior Art
This invention relates to methods and means for determining the presence of a ligand in a liquid medium based on the affinity of the ligand for a specific binding partner. In particular, this invention relates to methods and means for use in specific binding assays which do not employ radioactive materials.
Folate deficiences in the human body are a common cause of megaloblastic anemia. In humans, folic acid is metabolized to tetrahydrofolic acid and subsequently to 5-methyltetrahydrofolic acid (5'-mTHF). Often, the concentration of 5'-mTHF is measured using a competitive binding assay. It would be useful to include 5'-mTHF used in the standard reagents as a calibrator. Unfortunately, 5'-mTHF is very unstable and its use can require sealing the material in lyopholized form.
U.S. Pat. No. 4,350,659 to Riceberg of Corning Glass Works disclosed a process for stabilizing 5'-mTHF by complexing it with a binding protein such as folate binding protein (FBP). The complex is then frozen and lyophilized to yield a dry powder. Recommended storage of the powder includes air tight and light resistant containers. Lypholization permits storage of 5'-mTHF in a stable form until it is needed. Such techniques are impractical in the manufacture of assay kits and make it difficult to use in clinical laboratory settings. Moreover, after reconstituting the lypholized material, instability problems can reappear.
Deficiencies in vitamin B.sub.12 may result in neurological damage. Futhermore, as this vitamin is necessary for proper folic acid metabolism, its absence also results in megaloblastic anemias. Since megaloblastosis may also be produced by folate deficiency due to other causes, it is necessary to determine if the megaloblastosis is caused by a deficiency of either or both vitamins.
U.S. Pat. No. 4,399,228 to Riceberg of Corning Glass Works discloses a folate and vitamin B.sub.2 competitive protein binding assay. Radioactive .sup.57 Co or .sup.125 I tracer is added to patient samples and counted with a gamma counter. In the assay, the binding protein is covalently bound to porous glass. The endogenous binding proteins in the patient sample are destroyed by boiling the reaction tube. Because of the hazard and difficulty of handling radioactive materials, there have been many attempts to devise convenient specific binding assay systems which are as sensitive and rapid as radioimmunoassays but which utilize features other than radioactivity as the means for monitoring the binding reaction.
U.S. Pat. No. 4,028,465 to Lewin et al., of Bio-Rad Laboratories discloses a radioactive competitive assay procedure where sample serum folate is measured. The serum folate binding proteins are inactivated by heat. The invention discloses the use of a sulfhydryl such as dithiothreitol in buffer which can be used to stabilize folate prior to the heating process. The use of this stabilizer was advantageous over methods utilizing mercaptoethanol because it is an easily weighed solid with only a mild odor.
Folate and vitamin B.sub.12 assays typically employed heating or boiling steps prior to testing in order to liberate folate in the sample from endogenous binding proteins. The heating or boiling steps are difficult to accurately control and are time consuming. More recent assays denature samples by chemical means without boiling. One can achieve denaturation by using a strong base with or without other chemicals.
U.S. Pat. No. 4,418,151 to Forand et al., of Rohm and Haas Company also relates to a radioassay for serum folate. A measured amount of serum is mixed with a constant amount of radioactively tagged vitamin B12 and/or folate tracer. The solution is exposed to a mercaptan denaturing agent in the presence of a conversion agent such as potassium cyanide in a highly alkaline environment. The use of mercaptan solutions allows stabilization in the protecting buffer while high pH causes inactivation of the endogenous binding proteins.
U.S. Pat. No. 4,451,571 to Allen of University Patents teaches the use of strong base with a sulfhydral compound, such as betamercaptoethanol (BME), thioglycolate, thioglycerol or dithiothreitol (DTT). The sulfhydral compounds destroy endogenous binding proteins thereby liberating the sample vitamin B.sub.12 or folate to be measured. Although the strong base releases analyte from its binding protein, it does not substantially denature all endogenous binding protein. Therefore it is helpful to have another compound such as a sulfhydral to help liberate analyte from binding protein and also eliminate blocking antibodies which may interfere in the assay. The blocking antibodies can be troublesome to the assay because they react with binding factors.
U.S. Pat. No. 4,828,985 to Self of Cambridge Patent Developments teaches a method where secondary antibodies are raised against complexes of nonimmunogenic materials and primary antibodies against the nonimmunogenic materials. The secondary antibodies are not antibodies against either the nonimmunogenic materials nor the primary antibodies. Detection is accomplished by labelling the secondary antibodies with enzyme or some other detectable means.
The present invention is an improvement over existing technology in that it discloses a method that enables more coupling of specific binding proteins. This invention discloses a method whereby a mixture of two monoclonal or polyclonal antibodies or a mixture thereof against different epitopes of a binding protein gives increased coupling. This method can be utilized for several different assays in which analyte is detected. Another advantage of the present invention is that this multiclonal format allows the use of pteryolglutamic acid (PGA) as a calibrator in a folate assay instead of the unstable 5'-mTHF.